The complete nucleotide sequence ofthe genome of the type 1 poliovirus vaccine strain (LSc,2ab) was determined by using molecular cloning and rapid sequence analysis techniques. The restriction fragments of double-stranded cDNA synthesized from the vaccine strain RNA were inserted into the adequate sites of cloning vector pBR322. Sequence analysis of the cloned DNAs revealed that the virion RNA molecule was 7,441 nucleotides long and polyadenylylated at the 3' terminus. When the nucleotide sequence was compared with that of the genome of the virulent parental strain (Mahoney), 57 base substitutions were observed to be scattered all over the genome. Of these, 21 resulted in amino acid changes in a number of viral proteins. A cluster of amino acid changes is located in the viral coat proteins, especially in the NH2-terminal halfofthe viral capsid protein VP1.These results may imply that the mutations in the VP1 coding region contribute to attenuation.The genome ofpoliovirus is a single-stranded RNA with positive polarity, in which all of the viral genetic information is stored (1). This genomic RNA is composed of =7,500 nucleotides, polyadenylylated at the 3' terminus (2) and covalently attached to a genome-linked protein (VPg) at the 5' terminus (3-6). Re PVl(Sab)] is a live vaccine strain derived from the PV1(M) by spontaneous mutations during the attenuation process (9, 10).To determine the molecular basis for the biological differences between virulent and attenuated poliovirus strains, the sequences of large and unique RNase Ti-and A-resistant ohigonucleotides of PV1(M) and PV1(Sab) have been compared. We have shown that mutations detected by oligonucleotide analysis were caused by single base substitutions and appeared to be scattered all over the genome (11).For further comparative sequence studies, the restriction fragments obtained from double-stranded cDNA of the PV1(Sab) genome have been cloned (12). We report here the complete 7,441-nucleotide sequence of the PV1(Sab) genome, and the mutation sites were identified by comparison of our sequence with the known sequence of the PV1(M) genome (7,8).
Poliovirus infection of HeLa cells induces rapid shutoff of host protein synthesis, whereas translation of poliovirus RNA is not inhibited. It is presumed that shutoff is the result of proteolytic cleavage of component p220 of eucaryotic initiation factor 4F. To study whether poliovirus proteinase 2A is involved in this cleavage, we translated synthetic RNAs that contained the coding region for poliovirus-specific polypeptides P1 and 2A in vitro and assayed for cleavage of p220. We report here that cleavage of p220 occurred in all cases when active proteinase 2A was translated and that disruption of the coding sequence of 2A by linker insertion or deletion prevented processing of p220 in vitro. Activity of 2A was determined by its ability to cleave at the P1-P2 site of a segment of the poliovirus polyprotein. We also constructed a plasmid in which the 3'-most 500 nucleotides of the nontranslated region of encephalomyocarditis virus were linked to the coding sequence for poliovirus polypeptide 2A. Translation of the RNA transcript of this clone was very efficient and yielded a fusion protein that included 2A; this polypeptide also induced cleavage of p220. In vitro translation in the presence of antibodies against 2A specifically inhibited processing of p220, whereas incubation of in vitro translation products with antibodies against 2A after translation was completed did not prevent proteolysis of p220.
Plasmids have been constructed to generate substrates for the study of proteinases 2A and 3C of poliovirus. They contain the P1 (capsomer precursor) region of the poliovirus genome or P1 and part of P2 (a nonstructural precursor), which can be transcribed and translated in vitro. A transcript containing the entire 5' nontranslated region and the P1 region of the viral RNA gave poor translation in a reticulocyte translation system. Truncation of the 5' nontranslated region to its 3'-most segment gave acceptably good yields of radiolabeled P1. P1 was specifically processed to yield capsomer proteins by enzymes supplied in a postmitochondrial supernatant from poliovirus-infected cells. Thus, proteinase 3C can be supplied exogenously (in trans) and effect processing. This system may be used to provide P1 for the assay of proteinase 3C. Precursors that lacked either the 1A or 1D regions were poor substrates for proteinase 3C-observations that demonstrated a stringent structural requirement in processing by 3C. The translation product of a transcript encoding P1 and part of P2 was rapidly cleaved at the P1-P2 site in the absence of infected-cell extract. A transcript that contained a mutated 2A region gave a stable P1-P2 precursor that could be processed specifically by exogenous proteinase from infectedcell fractions. Processing of P1 appeared to require cleavage of the P1-P2 bond. These results support our previous data that 2A is the second polioviral proteinase and also provides a means of assaying proteinase 2A in vitro.Poliovirus genomic RNA is -7.5 kb in length (1) and is not conventionally "capped" but has an oligopeptide, VPg, of viral origin covalently attached to the 5' end. It has been shown that polysomal poliovirus RNA lacks VPg, and this is the only known difference between poliovirus mRNA and genomic RNA (ref. 2 and references therein).The RNA of poliovirus type 1 (Mahoney strain) encodes a single continuous reading frame of 2208 codons (1), which is typical of the family Picornaviridae, to which poliovirus belongs. In infected cells the translation product yields mature capsid and noncapsid proteins through proteolytic processing, largely by nonstructural viral protein 3C (3, 4), that cleaves the polyprotein precursor at 9 of 13 glutamineglycine pairs (ref. 5 and references therein). No proteins other than poliovirus-specific polypeptides are known to serve as substrates for proteinase 3C. Recently, we (6) presented evidence that viral protein 2A is also a proteinase and probably cleaves at two sites, both of which are tyrosineglycine pairs. Other workers have recently provided genetic evidence suggesting that proteinase 2A can act in trans in vivo and, hence, that it is a diffusible protein (7). It has also been speculated (8) that the final maturation cleavage of viral protein VPO is self-activated. Processing is to some extent cotranslational, insofar as the full-length translation product cannot be found under normal conditions in vivo (reviewed in ref. 9).The 5' nontranslated regions (NTR...
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